Opioid receptors, beyond their involvement in pain transmission, play a number of relevant physiological roles in the central nervous system and in peripheral organs. Opioid receptors can be considered a crossroads where endogenous opioid peptides and foreign opioids and opiates meet the cell and transmit their messages to another vast array of stimulus-response mechanism. In recent years, studies on their emerging roles have been favored by numerous and fruitful techniques that have opened new avenues of preclinical and clinical research that demands multidisciplinary approaches. The post-genomic era has opened up novel opportunities for the exploitation of these novel technologies. As an increasing number of investigators seek to harness the fruits of knowledge in these emerging fields, it is essential that well-tested protocols are made available to researchers. With this in mind, it is apposite to provide a collection of protocols to favor innovative studies on opioid receptors written by experts who are routinely employing these techniques in their laboratories. This book presents the protocols in the stepwise “cookbook” style of this well-known book series and summarizes of state-of-the art methods that have been utilized for understanding opioid receptor functionality at molecular, cellular, structural, and organism levels. Opioid Receptors: Methods and Protocols—Second Edition is, hence, an invaluable guide for researchers in the fields of neuroscience, biochemistry, pharmacology, and molecular biology.